Targeted amplification of selected polynucleotide sequences from a complex mixture of nucleic acids is of great interest in many areas, including genetic analysis, microorganism detection, diagnostics, environmental monitoring, and the like. Such amplification provides a number of advantages, including the conservation of scarce sample and the enrichment of sequences to be analyzed or for reducing overall sample complexity for improved analysis by “downstream” analytical techniques. Targeted amplification of multiple sequences has been carried out with conventional multiplex polymerase reaction (PCR), although this form of PCR is usually limited to amplifications of fewer then ten target sequences, e.g., Mackay, Clin. Microbiol. Infect., 10:190-212 (2004). Henegariu et al, Biotechniques, 23:504-511 (1997); Elnifro et al. Clin. Microbiol. Rev., 13:559-570 (2000); Gardner et al, J. Clin. Microbiol., 41: 2417-2427 (2003); Kimata et al, Microbiol. Immunol., 49:485-492 (2005); and the like. More recently, several techniques have been introduced to overcome some of the limitations of conventional multiplex PCR, particularly for simultaneously amplifying numbers of sequences in the range of from several tens to several hundreds, e.g. Dahl et al, Nucleic Acids Research 33: e71 (2005); Broude et al, Proc. Natl. Acad. Sci., 98:206-211 (2001); Shapero et al, Nucleic Acids Research, 32:e181 (2004); Faham et al, U.S. patent publ. 2003/0096291; Faham et al. U.S. patent publ. 2003/0104459; Zabeau et al. U.S. Pat. No. 6,045,994 and the like. However, in each of these methods, trade-offs have been made that make them unsuitable for many circumstances where multiplex amplifications is required. For example, most such methods rely on endonuclease digestion of a nucleic acid sample, which limit ones ability to target the sequences to be amplified, and some methods call for the use of extraordinary long primers, or other ancillary oligonucleotide components, for selecting fragments, that are difficult or expensive to synthesized on a routine basis.
In view of the limitations of current targeted amplification methods, it would be beneficial for many applications if such a method were available that neither relied on endonuclease digestion nor required difficult-to-synthesize components.